The mycotoxin contamination of feed results in billions of dollars of economic losses to animal husbandry world-wide and in some cases in health damage to human consumers due to transfer of contamination via dairy products, eggs and meats. The key mycotoxigenic moulds in partially dried grain are Penicillium verrucosum, producing ochratoxin (OTA) and Fusarium graminearum and F. sporotrichioides, producing deoxynivalenol (DON), nivalenol (NIV) and T-2 toxin in the damp cool climates of Northern Europe. In the South Aspergillus flavus is producing aflatoxins (AF), A. ochraceus—OTA and some Fusarium species are producing fumonisins (FUM) and trichothecenes DON and NIV (1, 2, 3).
Easy screening for mycotoxin contamination can be provided by a specialized Lab equipped with LC/MS, preferably with atmospheric pressure ionization (4). Up to 30 different toxins can be assayed in a single 30-min run (5).
Due to the diversity of mycotoxin chemical structures and properties, the mycotoxin binder solutions vary widely (6; 7; 8; 9; 10).
Mineral adsorbents were developed in the 1990-es, based on a number of silicates and alumo-silicates, such as phyllosilicate (11). They have been shown effective in suppressing the effect of 50 PPB Aflatoxin in broiler diets in terms of improving weight gain, FCR and mortality (12, 13). Efficiency of mineral adsorbents against Fumonisin and Zearalenone can vary from high (14) to limited. The efficiency is also limited against OTA (15; 16; 17, 18, 13). Against T-2 the efficiency is partial (18) and against trichlorocenes—such as DON and NIV—it is close to zero (19; 20; 21; 22; 23). On GI tract in-vitro model for healthy pigs a number of commercial mineral adsorbents were incapable of binding DON and NIV, while activated carbon provided substantial removal (24), however the later is known to render vitamins in feed less available as well.
Adsorption of non-aflatoxin mycotoxins can be significantly improved by coating mineral adsorbents with quaternary long-chain alkyl-aryl amines (25, 26, 27), a technique borrowed from HPLC, but such cationic surfactants are typically not allowed in animal feed.
Yeast culture addition has been known to improve health in dairy cows (28), poultry (29, 30) and pigs (31, 32), all species also addressed in a patent (33). Saccharomyces cerevisiae live yeast was shown to reduce the detrimental effects of aflatoxin in broiler diets (34).
However, due to multiplicity of mycotoxin structures and chemistry, the performance of yeast cells and cell wall mycotoxin binders has been demonstrated to not be universal (35). Zearalenone was shown to bind to the mannooligosaccharide fraction of the yeast cell wall at 75-80% at pH 3 and pH 4 at a ratio of 0.1 mg of toxin per gram of adsorbent (36; 37). Dietary MOS induced a significant reduction in liver cholesterol and liver fat levels on aflatoxin-stressed chickens (38). MOS also showed high binding capacity (76-87%, depending on toxin/binder ratio) for free Aflatoxin without feed (39), and for the toxin incorporated into poultry feed, the effective binder inclusion rate was demonstrated to be as low as 0.5 kg/ton (40). The binding of Aflatoxin to mannooligosaccharides was shown to be pH-dependent, pH 6.5 allowing more toxin binding (up to 80% versus 67%) compared to pH 4.5 (41).
U.S. Pat. No. 6,045,834, protecting the Mycosorb technology (42), claims that yeast cell wall and mineral clay are acting in synergy, providing better mycotoxin binding than yeast cell wall debris alone. In addition, the patent indicates that when the cell wall isolated from yeast is subjected to an alcohol shock (i.e. originating from anaerobic ethanol fermentations), it has a better binding capacity for mycotoxins (due to thickening and increase in surface area of the cell wall) than yeast cell wall material from an aerobic yeast fermentation. The patent demonstrates through in-vitro experiments that Aflatoxin, Zearalenone and Fumonisin were well bound by Mycosorb-type adsorbent from aqueous mixes with feed, but DON, OTA and T2 remained still in solution at 67-87% level of total content.
Another group was reporting similar results in vitro on toxin-contaminated broiler feed: modified manno-oligosaccharides (1 kg/ton) were binding 86% of aflatoxin B1 (@300 ppb), but only 28% of T-2 (@3000 ppb) and only 25% OTA (@2000 ppb), regardless of pH (43). Subsequent testing of yeast glucomannan demonstrated binding of 95% of total aflatoxins, 67% of FUM, 77% of ZEN, but only 33% of T-2, 13% of DON, 12% of OTA and 8% of NIV (44, 45).
On a GI tract in-vitro model for healthy pigs, yeast-based adsorbents, such as Mycosorb and its analogs, were incapable of binding trichothecenes DON and NIV, while treatment with activated carbon resulted in their substantial removal (24).
Yeast cell wall feed additives did not improve the feed intake, weight gain and feed conversion of 42-day broilers fed diets contaminated with 500 ppb OTA versus the birds on mycotoxin-free control diets (46, 47).
A glucan polymer product did not alleviate the toxic effects on mink consuming diets contaminated with fumonisin, ochratoxin, moniliformin, and zearalenone (48).
As a recent development, the DDGS from fuel ethanol industry may become an unexpected competitor to yeast cell wall-based mycotoxin binders, making them redundant. DDGS contains a significant amount of yeast cells, sufficient for binding aflatoxins and fumonisin, even taking into account their 3-fold concentration from maize grain to pot solids (49). The difficult to bind types of mycotoxins are also concentrated 3-fold, but cannot be alleviated by yeast-based DDGS components or specially added binders. Meanwhile, the negative effect of feeding DDGS with current mycotoxin levels to pigs only was calculated nationally at $2-8 million p.a. at current penetration of DDGS into swine feed and $30-290 million at inclusion of DDGS into all swine feed at 200 kg/ton (50).
The major part of distiller's grain is consumed by cattle. According to field observations, when DON concentrations were higher than 0.5 ppm, milk yield was reduced by 25 pounds (51). A maximum of 7.7 ppm and an average of 3.6 ppm of DON were reported for 54 samples of DDGS tested (accumulated crop years: May 1, 2000 through Apr. 30, 2007). The respective numbers for wet distiller's grain were 4.3 and 1.9 ppm (52). These numbers indicate that feeding DDGS and WDG to ruminants without control of DON already leads to substantial economic losses. Again, these losses cannot be alleviated using yeast cell wall-based mycotoxin binders.
The present invention is proposing to take advantage of the increased hydrophobicity of a number of ambivalent proteins in order to modify the surface of lignocellulosic material with a hydrophobizing layer. Without such modification, the adsorben surface remains rather hydrorophilic and characterized by low affinity and adsorption capacity towards hydrophobic mycotoxins. For example, endoglucanases of the cellulase complex of an industrial producer Trichoderma reesei were shown to possess abnormally high hydrophobicity and did not bind to ion exchange resins, but were retarded on a column for hydrophobic chromatography (53).
Microbial cellulases appear to be most suitable for the role of a protein modifying the lignocellulose surface towards increased hydrophobicity since they contain a special cellulose binding domain. This feature allowed the creation of cellulase enzyme products with targeted surface action and decreased damage of cotton fabrics by immobilizing the cellulase enzyme on cellulose particles (54).
The ambivalent nature of microbial cellulases plays a negative role during enzymatic stonewashing of jeans, an industry consuming a considerable part of commercially produced cellulase enzymes. When the enzyme molecule has a cellulose binding domain, the enzyme is strongly adsorbed on cotton, while the hydrophobic globule of the active site attracts particles of the washed off dye (55). The phenomenon is called backstaining, where the dye washed off from the denim is redeposited on the white fabric of pockets and denim areas of stone abrasion, both supposed ideally to be as white as possible. Despite a significant number of studies, use of microbial cellulases for hydrophobization of lignocellulose surfaces to create inexpensive and effective adsorbents has not been mentioned in the literature, nor in patents, and is a novelty proposed in the present invention. No other proteins are described in literature as surface modifiers of lignocellulose for these purposes.